Accumulating evidence suggests that a combined regimen of immunization, in which a non-viral vector such as a DNA vaccine is given, followed by a viral vector such as vaccinia virus, results in robust CTL and CD4 T cell responses in mice and non-human primates. An important area of induction of cellular immunity to HIV that has not been much studied is the ability to induce CTL and humoral responses at mucosal surfaces. Yet, since most exposure to HIV occurs via mucosal surfaces, a successful prophylactic vaccine against HIV is likely to require a mucosal component. In these studies, we are seeking to define prime/boost strategies for the induction of high levels of mucosal immunity to HIV envelope (env) glycoprotein. We propose to explore the possibility of using poly(DL-lactide-co-glycolide) (PLG) microparticles as vehicles for oral vaccination with plasmid DNA encoding gp160 in combination with recombinant viral vectors expressing the env gene products to enhance the antigen-specific mucosal and systemic immunity. The recombinant viruses used in these studies will include a replication-attenuated modified vaccinia virus Ankara (MVA), adeno-associated virus (AAV), and the E1/E3-deleted adenoviral (Ad) vector. The live vectors will be associated with liposomes for protection against gastric environment, increased transduction and vector dissemination in vivo. The overall goal of this effort is to derive ways to maximize the immunogenicity and safety of the HIV vaccine by inducing long-lasting protective immunity to the HIV env in mucosal and systemic sites. Furthermore, because systemic immunization with DNA plasmid or vaccinia virus does not induce antigen-specific immunity in mucosal tissues, the inductive sites of the mucosal immune system may still be naive to the live vectors delivered by oral vaccination. We hypothesize that oral vaccination with PLG-encapsulated DNA plasmid and liposome-associated non-replicating viral vectors expressing gp160 will augment the magnitude of immune responses to HIV env in mucosal and systemic tissues, and help to overcome the problem of preexisting immunity to the live vectors. The following specific aims will be pursued: i) to evaluate levels of env-specific cellular and humoral responses in systemic and mucosal tissues (Peyer's patches and lamina propria) of gastrointestinal track after oral vaccination with a) PLG-encapsulated DNA plasmid encoding gp160, b) liposome-associated recombinant viral vectors expressing gp160, and c) a combination of PLG-encapsulated DNA plasmid and liposome-associated viral vectors expressing the env glycoprotein; ii) to determine whether oral vaccination overcomes the barrier to recombinant vaccinia or adenovirus immunization caused by preexisting immunity to these pathogens, and whether multiple oral immunizations with liposome-associated AAV-env enhance gp160-specific immunity; and iii) to analyze levels of protection in mucosal tissues induced by the oral vaccination against intrarectal challenge with recombinant vaccinia virus expressing gp160.